Wound healing

ABSTRACT

A method of treating a chronic wound in a subject in need of such treatment is provided. The method includes administering to the subject at least one antioxidant agent in an amount effective to treat the wound. In some versions, the antioxidant agent is α-tocopherol or N-acetyl cysteine, or a combination of these compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of Provisional Patent ApplicationNo. 62/165,156, filed on May 21, 2015, which is incorporated byreference herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made in part with Government support under Grant No.R21A1078208 from the National Institutes of Health, National Instituteof Allergy and Infectious Diseases. The Government has certain rights inthis invention.

BACKGROUND

1. Field of the Invention

The invention relates to creating and treating wounds.

2. Related Art

Wound healing is a dynamic process involving many factors and cell typesincluding soluble mediators, blood cells, fibroblasts, endothelialcells, and extracellular matrix. Normal wound healing is divided intoseveral sequential phases that overlap in space and time: homeostasis,inflammation, granulation tissue formation, and tissue remodeling.Chronic wounds develop as a result of defective regulation of one ormore of the complex molecular and biological events involved in properhealing.

Chronic wounds in diabetics are one of the most common complications.Diabetic foot ulcers and other similar chronic wounds impact ˜6.5 Mpeople and cost ˜$25 B/year in the US alone. The critical need for acure of diabetic chronic wounds is underlined by the continuous increasein type II diabetes which accounts for 90-95% of all diabetes.Challenges in doing research and understanding these problematic woundsresult from varying disease etiologies, existing co-morbidities and,importantly difficulties with human tissue collection. For the mostpart, a clinician sees the patient when the wound is already at anadvanced stage of chronicity and critical evidence of causality isalready lost.

Oxidative and nitrosative stress that make up the redox environment havebeen at the epicenter of numerous diseases. Maintaining balance of theredox state in the body is a challenge and hence a variety of methodsand drugs have been used to meet this need for balance.

SUMMARY

In one aspect, a method of treating a wound in a subject in need of suchtreatment is provided. The method includes administering to the subjectat least one antioxidant agent in an amount effective to treat thewound. In some embodiments: a) the at least one antioxidant agentdecreases wound infection; b) the at least one antioxidant agentincreases the rate of wound healing; c) the at least one antioxidantagent is two or more antioxidant agents; d) the at least one antioxidantagent comprises a free radical scavenger, a lipid peroxidationinhibitor, or a combination thereof; e) the at least one antioxidantagent comprises N-acetyl cysteine, vitamin A, vitamin C, vitamin E,α-tocopherol, glutathione, lipoic acid, carotenes, coenzyme Q(ubiquinol), melatonin, ellagic acid, punicic acid, luteolin, catalase,superoxide dismutase, peroxiredoxins, cysteine, flavenoids, phenolics,or ergothioneine, or a physiological salt thereof, or any combinationthereof; f) the at least one antioxidant agent comprises α-tocopherol(α-TOC), N-acetyl cysteine, a physiological salt thereof, or anycombination thereof; g) the wound is a chronic wound; h) the wound is afrankly chronic wound; i) the wound contains exudate; j) the treatingcomprises dismantling of the biofilm, improvement in there-epithelialization of the wound, improvement in maturity of thegranulation tissue of the wound, decrease in wound infection, resolutionof inflammation, increase in rate of wound healing, establishment ofhomeostasis, or any combination thereof; k) the subject is a human oranimal; l) the subject is a diabetic; m) the wound is treated bydebridement before administering at least one antioxidant agent; n) orany combination of a)-m).

In some embodiments, the wound is a chronic wound. In these embodiments,the wound may or may not contain exudate. In some embodiments, the woundis a frankly chronic wound.

In another aspect, a method of making a chronic wound mouse model isprovided. The method includes creating a fresh wound in a db/db mouse,administering to the mouse one or more inhibitors of at least oneantioxidant agent, and allowing the fresh wound to develop into achronic wound. In some embodiments, a) the administering is performedbefore creating the fresh wound, after creating the fresh wound, orbefore and after creating the fresh wound; b) the inhibitors can be3-amino-1,2,4-triazole, mercaptosuccinic acid, or a combination thereof;c) the method further comprises covering the fresh wound with adressing, which can be a film-type dressing; d) the routes ofadministration for the one or more inhibitors can be intradermal,transdermal, parenteral, intravenous, intramuscular, intranasal,subcutaneous, percutaneous, intratracheal, intraperitoneal, topical,systemic, perfusion, lavage, direct injection, oral administration andformulation, or any combination thereof; e) the antioxidant agent isinvolved in wound healing; f) or any combination of a)-e).

In further aspects, a chronic wound mouse model and a method of usingthe mouse model to screen for agents that can treat and/or promotehealing of chronic wounds are provided.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present invention, reference isnow made to the following descriptions taken in conjunction with theaccompanying drawings, in which:

FIG. 1 is a simplified schematic illustration of the oxidative andnitrosative stress cycle.

FIG. 2 is a panel of graphs showing the levels of superoxide dismutase(SOD), H₂O₂, catalase (CAT) and glutathione peroxidase (GPx) afterwounding.

FIG. 3 is a schematic drawing of the oxidative and nitrosative stresscycle showing the effects of OH radicals.

FIG. 4 is a schematic drawing of a gain of ROS function experiment.

FIG. 5 is a panel showing results of inhibiting anti-oxidant enzymes.

FIG. 6 is a panel showing wound areas on the left graph and animalweight on the right graph.

FIG. 7 is a panel of graphs characterizing bacterial quantification andbiofilm formation. In FIG. 7A, Growth potential, the dashed linerepresents the levels of growth above which the bacterial have thecapacity to form biofilm. FIG. 7B shows the same bacteria growth onspecific media to identify the various species.

FIG. 8 is a panel of graphs characterizing bacterial quantification andbiofilm formation. The left graph shows bacteria growth on specificmedia to identify various species as in FIG. 7B. The right graph showsthe data of the left graph in a different configuration to more clearlyshow the bacteria competition during evolution of the biofilm formation.

FIG. 9 is a table of antibiotic resistance as the biofilm develops overtime.

FIG. 10 is a panel indicating the presence of bacteria in the tissues ofchronic wounds. In the left picture, small specks of green show thebacterial nuclei and large blobs show the cellular DNA. In the rightpicture, simultaneous staining with wheat germ agglutinin (WGA) showsclose association of the bacteria with the biofilm.

FIG. 11 is a panel of scanning electron micrographs indicating thepresence of bacteria in the tissues of chronic wounds. Both micrographsshow the biofilm is extensive on the surface of the wound.

FIG. 12 is a panel indicating that treatment with N-acetyl cysteine andα-tocopherol improves healing and weight gain. The photos illustrate theimprovement in healing after Antioxidant Agent (AOA) treatment. Thegraph on the left shows great improvement in size of the wound area andthe graph on the right shows weight recovery after AOA treatment.

FIG. 13 is a panel indicating that treatment with N-acetyl cysteine andα-tocopherol decreases oxidative stress. The graph on the left showsthat superoxide dismutase (SOD) is elevated as it tries to dismutatesuperoxide anions into H₂O₂. The graph on the right shows that H₂O₂ islower because the antioxidant enzyme activity is now elevated comparedwith the non-treated wounds (see FIG. 14).

FIG. 14 is a panel indicating that treatment with N-acetyl cysteine andα-tocopherol decreases oxidative stress. Both anti-oxidant enzymescatalase (left graph) and glutathione peroxidase (GPx; right graph) areelevated in the wounds treated with the antioxidant agents (AOA).

FIG. 15 is a panel indicating that treatment with N-acetyl cysteine andα-tocopherol decreases bacterial burden. The potential for biofilmformation is significantly decreased when the chronic wounds are treatedwith AOA (left graph) when compared to non-treated (right graph).

FIG. 16 is a panel indicating that treatment with N-acetyl cysteine andα-tocopherol decreases bacterial burden. Biofilm formation by theisolated species is significantly decreased when the chronic wounds aretreated with AOA (left graph) when compared to non-treated (rightgraph).

FIG. 17 is a table indicating that antibiotic resistance decreased asthe wounds are treated with AOA over time. This is the case for 3antibiotics used in the clinic.

FIG. 18 is a panel of scanning electron micrographs showing that thebiofilm is disappearing as the treatment continues (micrographs on theright compare with micrographs on the left).

FIG. 19 is a panel indicating that treatment with N-acetyl cysteine andα-tocopherol improves the quality of the healing tissue. The picturesindicate that the granulation tissue is much more mature in the woundstreated with both AOA (NAC and a-Toc).

FIG. 20 is a panel indicating that treatment with N-acetyl cysteine andα-tocopherol improves the quality of collagen deposition. The collagenfibers in the granulation tissue are much more mature in the woundstreated with both AOA (NAC and a-Toc).

FIG. 21 is a panel indicating that treatment with N-acetyl cysteine andα-tocopherol improves the quality of the epidermis. The epidermis ismuch more mature in the wounds treated with both AOA (NAC and a-Toc).

FIG. 22 is a schematic illustration of the framework for experiments.Creating chronic wound is shown on the left side of the schematic, andhealing chronic wounds is shown on the right side of the schematic.

DETAILED DESCRIPTION

In one aspect, a method of treating a wound in a subject is provided.The method includes administering to the subject at least oneantioxidant agent in an amount effective to treat the wound. In someembodiments, the wound can be a chronic wound. Chronic wounds include,but are not limited to, venous stasis ulcers, arterial ulcers, diabeticulcers, pressure ulcers, traumatic ulcers, post-surgical ulcers, smallvascular wounds, wounds with bacterial biofilm, and allergy-inducedwounds. Chronic wounds occur in diseases and conditions such as, but notlimited to, diabetes, vasculitis, ischemia, immune suppression, pyodermagangrenosum, fibrosis, edema, sickle cell anemia, and peripheralarterial disease atherosclerosis.

In some embodiments, the at least one antioxidant agent is oneantioxidant agent, two antioxidant agents, or more than two antioxidantagents. An antioxidant agent can be, but is not limited to, a freeradical scavenger or an inhibitor of lipid peroxidation, or acombination thereof.

Examples of antioxidant agents include, but are not limited to, N-acetylcysteine, vitamin A, vitamin C, vitamin E, glutathione, lipoic acid,carotenes, coenzyme Q (ubiquinol), melatonin, ellagic acid, punicicacid, luteolin, catalase, superoxide dismutase, peroxiredoxins,cysteine, flavenoids, phenolics, and ergothioneine, or a physiologicalsalt thereof, or a combination thereof. Any form of vitamin E can beused, including tocopherols such as α-tocopherol, and tocotrienols.

An antioxidant agent can be administered to a subject in various waysdepending, for example, on the particular antioxidant agent and the typeof wound. The routes of administration can include, e.g., intradermal,transdermal, parenteral, intravenous, intramuscular, intranasal,subcutaneous, percutaneous, intratracheal, intraperitoneal, topical,systemic, perfusion, lavage, direct injection, and oral administrationand formulation. In embodiments having multiple antioxidant agents, thedifferent agents can be administered by the same or different routes.

The length of administration will vary depending on, for example, theantioxidant agent or agents used, the subject being treated, thesubject's weight, the manner of administration and the judgment of theprescribing physician. For example, administration of the active agentor compound can occur for at least 20 days once a wound is chronic, oruntil the wound is visibly improved and on the way to healing.

In embodiments of the method, an antioxidant agent can be administeredin an amount effective to treat the wound, or when two or moreantioxidant are administered, each agent alone or a combination of theagents is in an amount effective to treat the wound. Thistherapeutically effective amount is an amount that results in animprovement or a desired change in the condition for which the agent orcombination is administered, whether the agent or combination isadministered once or over a period of time. For example, with respect tomethods of treating a wound, the improvement can be a dismantling of thebiofilm, improvement in the re-epithelialization of the wound,improvement in maturity of the granulation tissue of the wound, decreasein wound infection, resolution of inflammation, increase in rate ofwound healing, establishment of homeostasis, or any combination thereof.As is known, the therapeutically effective amount will vary dependingon, for example, the antioxidant agent or agents employed, the subjectbeing treated, the subject's weight, the manner of administration andthe judgment of the prescribing physician. With a diabetic subject, thelevels of agent in the blood and the health condition of the subject canalso be a consideration. In a mouse subject, for example, 200 mg/kg bodyweight of N-acetyl cysteine (equivalent to a 10% solution) and 50 mg/kgbody weight of α-tocopherol (equivalent to a 20% solution) can beadministered. Similar doses, appropriately scaled, can be used forhumans.

In some embodiments, the antioxidant agent can be administered as apharmaceutical composition. A pharmaceutical composition will typicallycontain a pharmaceutically acceptable carrier. Depending on the intendedmode of administration, the pharmaceutical compositions may be in theform of solid, semi-solid or liquid dosage forms, such as, for example,tablets, suppositories, pills, capsules, powders, liquids, suspensions,ointments or lotions, preferably in unit dosage form suitable for singleadministration of a precise dosage. The compositions may include aneffective amount of a selected compound in combination with apharmaceutically acceptable carrier and, in addition, may include otherpharmaceutical agents such as anti-viral agents, adjuvants, diluents,buffers, and the like. The compound may thus be administered in dosageformulations containing conventional non-toxic pharmaceuticallyacceptable carriers, adjuvants and vehicles.

For solid compositions, conventional nontoxic solid carriers include,for example, pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose,magnesium carbonate, and the like. Liquid pharmaceutically administrablecompositions may, for example, be prepared by dissolving, dispersing,etc., an active compound as described herein and optional pharmaceuticaladjuvants in an excipient, such as, for example, water, saline, aqueousdextrose, glycerol, ethanol, and the like, to thereby form a solution orsuspension. If desired, the pharmaceutical composition to beadministered may also contain minor amounts of nontoxic auxiliarysubstances such as wetting or emulsifying agents, pH buffering agentsand the like, for example, sodium acetate, sorbitan mono-laurate,triethanolamine acetate, triethanolamine oleate, etc. Actual methods ofpreparing such dosage forms are known, or will be apparent, to thoseskilled in this art. For oral administration, the composition willgenerally take the form of a tablet or capsule, or may be an aqueous ornonaqueous solution, suspension or syrup. Tablets and capsules for oraluse will generally include one or more commonly used carriers such aslactose and corn starch. Lubricating agents, such as magnesium stearate,are also typically added. When liquid suspensions are used, the activeagent may be combined with emulsifying and suspending agents. Ifdesired, flavoring, coloring and/or sweetening agents may be added aswell. Other optional components for incorporation into an oralformulation herein include, but are not limited to, preservatives,suspending agents, thickening agents, and the like. One skilled in thisart may further formulate the compound in an appropriate manner, and inaccordance with accepted practices, such as those disclosed inRemington's Pharmaceutical Sciences, Gennaro, Ed., Mack Publishing Co.,Easton, Pa. 1990.

A salt of an antioxidant agent can be a physiologically acceptable salt,which can be a pharmaceutically acceptable salt. Physiologicallyacceptable salts and pharmaceutically acceptable salts are well known inthe art and include salts prepared from pharmaceutically acceptablenon-toxic acids, including inorganic acids and organic acids. Suitablenon-toxic acids include inorganic and organic acids such as acetic,benzenesulfonic, benzoic, camphorsulfonic, citric, ethenesulfonic,fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic,lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic,pantothenic, phosphoric, succinic, sulfuric, tartaric acid,p-toluenesulfonic acids, and the like. Salts formed with, for example, afree carboxy group of an amino acid residue or a peptide, can be derivedfrom inorganic bases including, but not limited to, sodium, potassium,ammonium, calcium or ferric hydroxides, and organic bases including, butnot limited to, isopropylamine, trimethylamine, histidine, and procaine.

The subject can be a human or another animal, such as another mammal.

The present invention may be better understood by referring to theaccompanying examples, which are intended for illustration purposes onlyand should not in any sense be construed as limiting the scope of theinvention.

EXAMPLE 1

The methods in this Example were described in Sandeep Dhall, Danh C. Do,Monika Garcia, et al., “Generating and Reversing Chronic Wounds inDiabetic Mice by Manipulating Wound Redox Parameters,” Journal ofDiabetes Research, vol. 2014, Article ID 562625, 18 pages, 2014.doi:10.1155/2014/562625, incorporated by reference herein.

Dermal Excision Wound Model, Preparation of Tissue for Extracts and forHistology

The surgical procedures discussed here were approved by theInstitutional Animal Care and Use Committee of the University ofCalifornia, Riverside. Buprenex and ATZ are administered 30 and 20minutes before surgery, respectively. The mouse is then placed in anenclosed container that is hooked up to a isoflurane vaporizer in thechemical hood. Once the mouse is knocked out or no longer moving, it isplaced on a white surgical pad and the head is fitted with a nose conethat is secured to allow continuous administration of isoflurane for theduration of the surgery. A wipe (Kimwipe) sprayed with 70% ethanol or aclinical ethanol towelette is used to lightly wipe the area where thewound will be made (to minimize killing of the normal bacterial presentin the skin). This also provides a clean surface for the tegaderm tostick well since dust from the bedding, food, or skin can prevent thetegaderm from sticking properly. Full thickness 7 mm punch wounds(excision of the skin and the underlying panniculus carnosus) were madeon the back of the mice. The animals were euthanized usingcarbon-dioxide at various time points and the wound tissue was collectedfor histology and protein analysis using a 10 mm punch (woundbed+surrounding tissue). For protein analysis, zirconium oxide beadsweighing approximately the same as the wound tissue were added to tissuein safe lock tubes, followed by addition of 10 μL of RIPA buffer per mgof tissue. The tissues were bullet blended for homogenization. Theextracts were then centrifuged at 14000 rpm for 15 minutes at 4° C. Thesupernatants were used fresh or aliquots prepared and stored at −80° C.for later use. The samples were normalized to protein levels.

Superoxide Dismutase Activity Assay

Total tissue superoxide dismutase (SOD) activity was measured by using acommercially available kit (Cayman Chemical, Catalog number 706002, AnnArbor, USA) that measures all three types of SOD (Cu/Zn-, Mn-, andEC-SOD). One unit of SOD is defined as the amount of enzyme needed tocause 50% dismutation of the superoxide radical. Briefly, xanthineoxidase and hypoxanthine generate superoxide radicals that aredismutated by SOD and in the process tetrazolium salt are converted to aformazan dye that is read at 450 nm. The SOD activity of the samples wascalculated from the linear regression of a standard curve that wasdetermined using the SOD activity of bovine erythrocytes at variousconcentrations run under the same conditions. The SOD activity wasexpressed as U/mL of tissue extract.

Hydrogen Peroxide Activity Assay

Tissue hydrogen peroxide (H₂O₂) levels were measured by using acommercially available kit (Cell Technology Inc., Catalog number FLOH100-3, Mountain View, USA) that utilizes a nonfluorescent detectionreagent. The assay is based on the peroxidase-catalyzed oxidation byH₂O₂ of the nonfluorescent substrate 10-acetyl-3,7-dihydroxyphenoxazineto a fluorescent resorufin. 50 μL of tissue extracts collected atdifferent time points after wounding and normalized to proteinconcentration was mixed with 50 μL of the reaction cocktail in an opaque96-well assay plate. Fluorescent intensities were measured at 530 nm(excitation)/590 nm (emission) using a Victor 2 (fluorescence andabsorbance) microplate reader. The amounts of H₂O₂ in the supernatantswere derived from a seven-point standard curve generated with knownconcentrations of H₂O₂.

Catalase Activity Assay

Tissue catalase activity was measured by using a commercially availablekit (Cayman Chemical, Catalog number 707002, Ann Arbor, USA). The enzymeassay for catalase is based on the peroxidatic function of catalase withmethanol to produce formaldehyde in the presence of an optimalconcentration of H₂O₂. The formaldehyde produced was measuredspectrophotometrically, with4-amino-3-hydrazino-5-mercapto-1,2,4-triazole (purpald) as thechromogen, at 540 nm in a 96-well place. The catalase activity wasexpressed as nmol/min/mL of tissue extract.

Glutathione Peroxidase Activity Assay

A commercially available kit (Cayman Chemical, Catalog number 703102,Ann Arbor, USA) was used to measure tissue glutathione peroxidase (GPx)activity. The activity was measured indirectly by a coupled reactionwith glutathione reductase (GR). GPx reduces H₂O₂ to H₂O and in theprocess oxidized glutathione (GSSG) is produced that in turn is recycledto its reduced state by GR and NADPH. Oxidation of NADPH to NADP+ isaccompanied by a decrease in absorbance at 340 nm. Under conditions inwhich GPx activity is rate limiting, the rate of decrease in theabsorbance measured at 340 nm, in a 96-well plate at 1 min interval fora total of 5 min using a Victor 2 microplate reader, is directlyproportional to the GPx activity of the sample. GPx activity wasexpressed as nmol/min/mL of tissue extract.

Chronic Wound Model

To generate chronic wounds in db/db mice we performed full thickness 7mm diameter excision wounds on the dorsum of 6-7-month-old mice. Twentyminutes prior to wounding, mice were treated once intraperitoneally (IP)with 3-amino-1,2,4-triazole (ATZ) (Aldrich Chemistry; St. Louis, Mo.) at1 g/kg body weight, an inhibitor for catalase. Immediately afterwounding, they were treated once topically with the inhibitor for GPx,mercaptosuccinic acid (MSA), (Sigma Lifesciences; St. Louis, Mo.) at 150mg/kg body weight. Immediately after wounding, the wounds were coveredwith tegaderm (3 M; St. Paul, Minn.) to prevent contamination and werekept covered for the duration of the experiments. In these mice it iseasy to fully remove the hair from the back and hair grows very slowly;hence we had no problems keeping the tegaderm in place. The tegaderm wasremoved periodically to take pictures of the wound and then immediatelyreplaced. The wounds were fully chronic 20 days after wounding andremained open sometimes for more than 3 months, depending on theexperiment. Control db/db mice were treated exactly the same way butinstead of inhibitors of the antioxidant enzymes they were treated withthe vehicle (PBS). To reverse chronicity, at 20 days, the antioxidantNAC (Aldrich Chemistry (St. Louis, Mo.)) was topically applied to thewound at 200 mg/kg and the tegaderm replaced. Simultaneously, the micewere injected intraperitoneally with α-toc, Sigma Lifesciences (St.Louis, Mo.) at 50 mg/kg. This treatment continued with NAC applied tothe wound topically every day using an insulin syringe to deposit thesolution under the tegaderm and over the wound and with α-toc IP everyother day for 20 days (40 days after wounding). At this point, theantioxidant treatment was stopped and the wounds went on to heal around30 days after initiation of treatment with antioxidants (50 days afterwounding). For the antioxidant controls, the mice were treated exactlythe same but with vehicle rather than antioxidants. In some experiments,tissues were collected at various time points for detailedhistological/histochemical, biochemical, and cellular/molecularevaluation, and in some cases the tissues were analyzed for type andlevel of bacterial infection/biofilm production. Chronic wounds weresuccessfully created in over 100 animals

Bacteria Isolation and Characterization

To obtain the wound microbiome samples we used sterile cotton Q-tips toswab the wound bed, including the surface of the wound, but yetminimizing disruption of the wound microenvironment to allow forlongitudinal studies of the microbiome. The content of each swab wassuspended in 1.0% w/v protease peptone and 20.0% v/v glycerol solution.Wounded tissue for bacteria analysis was obtained using sterile scissorsand suspended in 1.0% w/v protease peptone and 20.0% v/v glycerolsolution. Tissues were homogenized in the presence of zirconium oxidebeads using a bullet blender at 4° C. Bacteria were cultured for 16-18 hat 37° C. on tryptic soy agar plates (BD Difco, Sparks, Md.), containing5.0% v/v defibrinated sheep blood (Colorado Serum Company, Denver,Colo.), and 0.08% w/v Congo red dye (Aldrich Chemistry, St. Louis, Mo.).Colonies were differentiated and isolated based on size, hemolyticpattern, and Congo red uptake. Resulting cultures were examined usingGram stain and visualized with optical microscopy. Gram-negative rodswere characterized using the API20E identification kit (Biomerieux,Durham, N.C.) and oxidase test (Fluka Analytical, St. Louis, Mo.). Whenrequired, the Pseudomonas Isolation Agar culture test, 42° C. growthtest in tryptic soy broth (TSB) (BD Difco, Sparks, Md.), and motilitytest were used. Gram positive cocci cultures were differentiated basedon catalase activity and coagulation activity (Fluka Analytical, St.Louis, Mo.), 6.5% w/v NaCl tolerance test, and hemolytic activity.Biofilm production was quantified using methods described previouslywith minor modifications. Briefly, 3-5 μL of the wound swabbed samplewas seeded in 100 μL of TSB and grown in humidified incubator at 37° C.in a 96-well polystyrene flat bottomed tissue culture plate under staticcondition. Bacterial content was removed by inverting and gentlyflicking the plate. The plate was washed three times by slowlysubmerging the plate and gently flicking the inverted plate to removethe water. The wells were dried by tapping onto absorbent paper and thenair dried at 65° C. for 30 minutes. The plate was cooled and stainedwith Hucker crystal violet for 5 minutes. Excessive stain was removed byrinsing the plate with water and then air dried overnight. The opticaldensity at 570 nm was then taken using the SpectraMax M2e microplatereader (Molecular Device, Sunnyvale, Calif.). Samples that give an OD ofor above 0.125 were considered biofilm positive whereas OD below 0.125was considered biofilm negative.

Viable Bacteria Cells Count

Wound swab samples were resuspended in sterile Luria broth (LB) to yielda 1:4 v/v ratio of sample-to-TSB solution. Bacterial colonies werevisually counted on trypticase soy agar plates containing 5% sheep redblood cells incubated at 37° C. overnight in a humidified incubator.

Community Minimal Inhibitory Concentrations Assay

Wound swab samples (containing bacteria) that were seeded on flatbottomed tissue culture plates for 3-4 hr at 37° C. in a humidifiedincubator were challenged with antibiotic for 12 hr at variousconcentrations in TSB. Optical density at 595 nm (OD 595 nm) was used toquantify bacterial growth. The community minimal inhibitoryconcentration (CMIC) is defined as the lowest concentration ofantibiotic that resulted in ≦50% increase in OD 595 nm compared to thatbefore introduction of antibiotic.

Bacterial Staining

Frozen sections of chronic wound tissues were stained using ViaGramRed+Bacterial Gram-Stain and Viability Kit (Life Technologies, Carlsbad,Calif.) with modifications to the manufacturer's protocol. Briefly, thefrozen tissue sections were washed in 1× PBS for 5 minutes at roomtemperature to remove the OCT. Sections were then incubated in wheatgerm agglutinin (WGA) conjugate stock solution for 5 minutes. The WGAsolution was drained off the slide followed by the addition of 2.5 μL ofthe DAPI/Sytox Green working solution for 10 minutes at roomtemperature. The excess working solution was then removed from thesection. Sections were mounted and visualized using a NikonMicrophot-FXA microscope with a Nikon DS-Fi1 digital camera.

Scanning Electron Microscopy

Tissues collected were fixed in 4% paraformaldehyde for 4 hrs at roomtemperature and then processed as described previously. Briefly, sampleswere dehydrated in a series of ethanol for 20 min each followed bycritical point drying of the tissues, using Balzers CPD0202 and Au/Pdsputtering in the Sputter coater Cressington 108 auto. The samples wereimaged using an XL30 FEG scanning electron microscope.

Biofilm Carbohydrate Composition

Chronic wound swab samples were washed with 80% and 100% ethanol toeliminate low molecular weight components and then with 2:1 (v/v)chloroform:methanol to remove lipids and acetone in preparation fordrying. Samples were desiccated under vacuum in the presence of P₂O₅.Total protein content of the dried swab sample was estimated by theLowry protein assay. Total carbohydrate content of dried swab sample wasestimated by colorimetric phenol-sulfuric acid assay, using gum arabicas the standard. For glycosyl composition analysis, dried swab samplewas cleaved by trifluoroacetic acid hydrolysis and the resultingmonosaccharides were derivatized by methanolysis, N-acetylation, andtrimethylsilylation as described previously, with minor modifications.Gas chromatography-flame ionization detection and gaschromatography-mass spectrometry were performed as previously described.DNA was extracted using the DNeasy Blood and Tissue kit (Qiagen,Chatsworth, Calif.) according to the manufacturer's instructions. DNAconcentration was measured and purity confirmed by calculating the OD260/OD 280 absorption ratio.

Second Harmonic Generation (SHG) Imaging

SHG imaging was done as previously described. Equipped with an NLOinterface for a femtosecond Titanium-Sapphire laser excitation source(Chameleon-Ultra, Coherent, Incorporated, Santa Clara, Calif.) formultiphoton excitation, an inverted Zeiss LSM 510 NLO META laserscanning microscope (Carl Zeiss Microscopy, LLC, Thornwood, N.Y.) fortransmitted light and epifluorescence was used. The Chameleon laserprovided femtosecond pulses at a repetition rate of about 80 MHz, withthe center frequency tunable from 690 to 1040 nm. A long workingdistance objective (Zeiss, 40× water, N.A. 0.8) was used to acquireimages. The sample two-photon signals were epicollected anddiscriminated by the short pass 650 nm dichroic beam splitter. A METAdetection module with signals sampled in a 394-405 nm detection range(lex=800 nm) was used to collect the SHG images. Each image presented inthis work is 12 bit, 512×512 pixels representing 225 mm×225 mm field ofview.

Statistical Analysis

We used Graphpad Instat Software and Sigmaplot Software. Analysis ofvariance (ANOVA) was used to test significance of group differencesbetween two or more groups. In experiments with only two groups, we usedthe Student's -test. Because the differences we observe are not small,we perform experiments in groups of three mice and then repeat theexperiment in groups of three as many times as needed to be confident ofthe results. For the majority of the cases, 2 sets of experiments to atotal of 6 animals were sufficient to achieve significant results.

EXAMPLE 2

This Example shows whether early reduction in the antioxidant capacityimmediately after wounding leads to the development of chronic woundsand whether application of antioxidants to these chronic ulcers reducesthe oxidative stress and leads to restoration of normal healing.

I. Excessive Redox Environment in Impaired Wounds of the Diabetic MouseModel, db/db^(−/−), Mimics the Environment Present in Human ChronicWounds

Understanding how wounds become chronic will provide insights to reversechronicity. We hypothesized that the high levels of oxidative stress(OS) in wound tissue is a critical component for generation ofchronicity. To test this possibility we used the db/db^(−/−) model ofimpaired healing and tested for key molecules involved in oxidativestress during wound healing (data are shown in the Figures). FIG. 1shows a simplified version of the ROS/NOS cycle. We examined the levelsof the molecules depicted in the boxes (FIG. 2). As early as 4 hrs afterwounding, we showed that SOD (superoxide dismutase) is elevated as ittries to dismutate the superoxide anions (O₂ ^(·−)) to H₂O₂ which isalso highly elevated (FIG. 2), and much like in humans, those levelsincrease with age. In addition, we also show that the two most potentantioxidant enzymes present in the wound, catalase and glutathioneperoxidase (GPx), which process H₂O₂ to H₂O and O₂, are down regulatedor not changed, respectively, resulting in the building of H₂O₂ in thewound tissue (FIG. 2). The increased H₂O₂ can react with Fe²⁺ ions toproduce *OH radicals which are a highly damaging molecule to proteins,lipids and DNA (FIG. 3). Together these data show high levels ofReactive Oxidative Species (ROS) in the wound.

II. Manipulation of Specific Redox Parameters in the Wound Environmentcan Create and Reverse Chronic Wounds

To show that these ROS are critical for the development of chronicwounds we perform gain and loss of function experiments. For the gain offunction experiments, we further increased ROS in the wound tissue byinhibiting the Catalase and GPx, the two powerful anti-oxidant enzymesmentioned above, using specific inhibitors for their activity. At thetime of wounding, catalase was inhibited with 3-Amino-1,2,4-triazole(ATZ) and GPx with mercaptosuccinic acid (MSA) (FIG. 4). Right aftertreatment the wound were covered with tegaderm a dressing approve forhuman use. In the figures these are called IAE (inhibitor ofanti-oxidant enzymes). For details of the methodology see Dhall et al.,2014 (Dhall et al., Journal of Diabetes Research, Volume 2014, ArticleID 562625) and Example 1 above. This treatment applied at the time ofwounding was necessary and sufficient to cause the wounds to becomechronic producing exudate and biofilm whereas treatment with PBS, usedas a vehicle for the delivery of the enzyme inhibitors, heal in thenormal time period for these mice (FIG. 5). The wound area enlarges andthe mice lose weight (FIG. 6). The biofilm develops with time withoutany manipulation on our part and is composed of many different bacteriathat have been found in human chronic wounds (FIG. 7,8). With time thevarious biofilm forming bacteria compete with each other and in the endone, E. cloacae, wins. Presence of biofilm was confirmed by antibiotictreatment, and microscopy. The former showed clearly that at day 4 whenthere is no biofilm forming capacity (FIG. 7) the antibiotic dose neededto eliminate the bacteria is low but that increases dramatically overtime as the biofilm forms (FIG. 9). For the microscopic observations weuse sytox green to visualize the bacteria I the tissue and wheat germagglutinin to see the biofilm matrix (FIG. 10). We also used scanningelectron microscopy to visualize the biofilm on the surface of the wound(FIG. 11).

To perform the loss of ROS function we treated the wounds withantioxidant agents (AOA). We chose to use N-Acetylcysteine (NAC), anamino acid that is critical for the function of GPx and also breaks downmucus and α-tocopherol (α-Toc) a specific form of Vit E that is a verypotent inhibitor of lipid peroxidation. To reverse chronicity, at 20days when the wounds are considered frankly chronic in this mouse modelsystem, we treated the wounds with NAC topically followed by replacementof the Tegaderm. Simultaneously, the mice were injectedintraperitoneally with α-Toc. This treatment continued with NAC appliedto the wound topically every day using an insulin syringe to deposit thesolution under the Tegarderm and over the wound and with a-Toc IPevery-other-day for 20 days (40 days post-wounding). At this point, theantioxidant treatment was stopped and the wounds went on to heal around30 days after initiation of treatment with antioxidants (50 dayspost-wounding) (FIG. 12). The oxidative stress was reversed, the biofilmdismantled (FIGS. 13-18) and the quality of the tissue was much improvedas detected by histology. We used hematoxylin an eosin staining forgeneral histology (FIG. 19), and Masson-thriochrome and second harmonicgeneration imaging (SHIM) to examine the quality of the collagen fiberin the granulation (healing) tissue (FIG. 20). We also usedimmunolabeling to show that the quality of the epidermis was muchsuperior when we used treated with NAC and α-Toc (FIG. 21). For theantioxidant controls, the mice were treated exactly the same way butwith vehicle rather than antioxidants.

III. Summary

Inhibition of antioxidant enzymes catalase and GPx in db/db mice uponwounding leads to development of chronic wounds.

Bacteria are observed in db/db mouse chronic wounds as early as 4 dayspost-wounding, even in the absence of bacterial inoculation.

Impaired healing can be restored by treatment with antioxidants NAC anda-Tocopherol.

Imbalanced levels of oxidative stress markers (SOD, H2O2, GPx andCatalase), dismantling of the biofilm and improved granulation tissuecan be restored with antioxidant treatment.

Dermis and epidermis development during healing is significantlyimproved by treatment with antioxidants.

The framework for the study is shown in FIG. 22.

CONCLUSIONS

Creation of redox imbalance in the wound microenvironment shortly afterwounding in db/db mice leads to the development of chronic wounds withincrease in oxidative stress, the presence of biofilm-forming bacteriaand high levels of tissue damage.

These effects can be reversed by application of antioxidant agents,leading to reduced oxidative stress, reduced presence of bacteria andimproved tissue quality.

Our findings demonstrate the important role of redox balance at theearly stages of healing and suggest that treating chronic wounds bydebridement followed by treatment of antioxidant agents should lead tosignificantly improved healing.

We have identified a major underlying trigger mechanism of poor healingthat occurs shortly after injury. The implication of these results isthat application of antioxidants to chronic wounds after debridementwould lead to significantly improved healing.

REFERENCES

The following publications are incorporated by reference herein in theirentirety:

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Although the present invention has been described in connection with thepreferred embodiments, it is to be understood that modifications andvariations may be utilized without departing from the principles andscope of the invention, as those skilled in the art will readilyunderstand. Accordingly, such modifications may be practiced within thescope of the invention and the following claims.

What is claimed is:
 1. A method of treating a chronic wound in a subjectin need thereof, comprising administering to the subject at least oneantioxidant agent in an amount effective to treat the wound.
 2. Themethod of claim 1, wherein the at least one antioxidant agent is two ormore antioxidant agents.
 3. The method of claim 1, wherein the at leastone antioxidant agent is a free radical scavenger, a lipid peroxidationinhibitor, or a combination thereof.
 4. The method of claim 1, whereinthe at least one antioxidant agent comprises N-acetyl cysteine, vitaminA, vitamin C, vitamin E, glutathione, lipoic acid, carotenes, coenzyme Q(ubiquinol), melatonin, ellagic acid, punicic acid, luteolin, catalase,superoxide dismutase, peroxiredoxins, cysteine, flavenoids, phenolics,ergothioneine, a physiological salt thereof, or any combination thereof.5. The method of claim 1, wherein the at least one antioxidant agentcomprises α-tocopherol, N-acetyl cysteine, a physiological salt thereof,or any combination thereof.
 6. The method of claim 1, wherein thechronic wound is a frankly chronic wound.
 7. The method of claim 6,wherein the frankly chronic wound contains exudate.
 8. The method ofclaim 1, wherein the treating comprises dismantling of the biofilm,improvement in the re-epithelialization of the wound, improvement inmaturity of the granulation tissue of the wound, decrease in woundinfection, resolution of inflammation, increase in rate of woundhealing, establishment of homeostasis, or any combination thereof. 9.The method of claim 1, wherein the subject is a human or animal.
 10. Themethod of claim 9, wherein the subject is a diabetic.
 11. The method ofclaim 1, wherein the wound is treated by debridement beforeadministering at least one antioxidant agent.